Summary Blood coagulation factor VIII is a protein cofactor that is essential for the proper regulation of the clotting cascade. Lack of factor VIII function causes hemophilia A, the most common genetic bleeding disorder, which affects 1 in 5,000 males worldwide. Treatment for hemophilia A consists of therapeutic infusions of a functional form of factor VIII, termed ?replacement therapy.? Complications arise, however, whereby replacement therapy commonly results in the development of an inhibitory immune response, affecting 25-30% of hemophilia patients receiving treatment. By structurally characterizing the nature by which factor VIII forms different complexes in circulation, such as with activated platelet surfaces, von Willebrand factor multimers and inhibitory antibodies, a better understanding for how to engineer factor VIII to overcome the immune response and remain highly active may be achieved. In our previous studies, we have structurally characterized ?classical? and ?non-classical? inhibitory antibodies that target the factor VIII C2 domain, which have resulted in further understanding of the anti- C2 immune response. We?ve also developed a working model for how factor VIII binds to activated platelet surfaces based on our previous structural and mutagenesis data. To further test our working model for membrane binding, this proposal will initially focus on determining the X-ray crystal structure of the factor VIII C2 domain bound to the soluble headgroup of phosphatidylserine and generating hemophilia A-associated mutants of the factor VIII C2 domain at Arg2320. To further aid in the advancement of superior factor VIII therapeutic molecules for gene therapy, we will also determine a high resolution X-ray crystal structure of a B domain-deleted human/porcine chimeric construct of factor VIII that is currently being developed as an engineered therapeutic protein with high biosynthetic activity. Furthermore, we will study different factor VIII protein complexes that arise in circulation, namely with its circulatory partner, von Willebrand factor, as well as with anti-C1 domain inhibitory antibodies associated with hemophilia A treatment. Factor VIII is known to bind tightly to the D?D3 domains of VWF. Currently, we are able to generate high levels of expression for the D? or D?D3 domains, which should be amenable to structural studies. Lastly, we will optimize the expression and purification of C1 domain-containing fVIII constructs for structural studies with anti-C1 domain inhibitory antibodies. By examining the factor VIII immune response at atomic resolution, paired with structural characterization of factor VIII circulatory complexes, our results will illustrate in detail the life cycle of factor VIII and its procoagulant activity. The structural data that result from this study will assist in the engineering and development of the next generation therapeutics to better treat hemophilia A patients worldwide.